Tumor associated peptide and uses thereof

ABSTRACT

The invention relates to a peptide with the sequence of RLLEFYLAM, methods for the use of the peptide, and antisera and monoclonal antibodies against the peptide. The peptide is derived from the NY-ESO-1 molecule, which form complexes with HLA molecules, leading to lysis of cells presenting these complexes, by cytolytic T lymphocytes.

RELATED APPLICATION

[0001] This application is a continuation in part of application Ser.No. 09/344,040, filed Jun. 25, 1999, which is a continuation in part ofSer. No. 09/105,839, filed Jun. 26, 1998, which is itself a continuationin part of application Ser. No. 08/851,130 filed on May 5, 1997. All ofthese Applications are incorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to the isolation and cloning of geneswhich are members of the “SSX” family, which is discussed herein, andthe uses thereof, including determination of cancer. Also a part of theinvention are peptides derived from these SSX genes, as well as from theNY-ESO-1 gene. These peptides stimulate proliferation of cytolytic Tcells, and thus are useful as markers for presence of disorders such ascancer, for HLA-A2 cells, and as therapeutic agents for treating cancer.

BACKGROUND AND PRIOR ART

[0003] It is fairly well established that many pathological conditions,such as infections, cancer, autoimmune disorders, etc., arecharacterized by the inappropriate expression of certain molecules.These molecules thus serve as “markers” for a particular pathological orabnormal condition. Apart from their use as diagnostic “targets,” i.e.,materials to be identified to diagnose these abnormal conditions, themolecules serve as reagents which can be used to generate diagnosticand/or therapeutic agents. A by no means limiting example of this is theuse of cancer markers to produce antibodies specific to a particularmarker. Yet another non-limiting example is the use of a peptide whichcomplexes with an MHC molecule, to generate cytolytic T cells againstabnormal cells.

[0004] Preparation of such materials, of course, presupposes a source ofthe reagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

[0005] To date, two strategies have been employed for the detection ofsuch antigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines. Transfectants arescreened for the expression of tumor antigens via their ability toprovoke reactions by anti-tumor cytolytic T cell clones. The biochemicalapproach, exemplified by, e.g., Mandelboim, et al., Nature 369: 69(1994) incorporated by reference, is based on acidic elution of peptideswhich have bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

[0006] These two approaches to the molecular definition of antigens havethe following disadvantages: first, they are enormously cumbersome,time-consuming and expensive; second, they depend on the establishmentof cytotoxic T cell lines (CTLs) with predefined specificity; and third,their relevance in vivo for the course of the pathology of disease inquestion has not been proven, as the respective CTLs can be obtained notonly from patients with the respective disease, but also from healthyindividuals, depending on their T cell repertoire.

[0007] The problems inherent to the two known approaches for theidentification and molecular definition of antigens are bestdemonstrated by the fact that both methods have, so far, succeeded indefining only very few new antigens in human tumors. See, e.g., van derBruggen et al., Science 254: 1643-1647 (1991); Brichard et al., J. Exp.Med. 178: 489-495 (1993); Coulie, et al., J. Exp. Med. 180: 35-42(1994); Kawakami, et al., Proc. Natl. Acad. Sci. USA 91: 3515-3519(1994).

[0008] Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer associatedantigens.

[0009] One key methodology is described by Sahin, et al., Proc. Natl.Acad. Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also,see U.S. patent application Ser. No. 08/580,980, and filed on Jan. 3,1996, and U.S. Pat. No. 5,698,396. All three of these references areincorporated by reference. To summarize, the method involves theexpression of cDNA libraries in a prokaryotic host. (The libraries aresecured from a tumor sample). The expressed libraries are thenimmunoscreened with absorbed and diluted sera, in order to detect thoseantigens which elicit high titer humoral responses. This methodology isknown as the SEREX method (“Serological identification of antigens byRecombinant Expression Cloning”). The methodology has been employed toconfirm expression of previously identified tumor associated antigens,as well as to detect new ones. See the above referenced patentapplications and Sahin, et al., supra as well as Crew, et al., EMBO J144: 2333-2340 (1995).

[0010] The SEREX methodology has been applied to esophageal cancersamples, and an esophageal cancer associated antigen has now beenidentified, and its encoding nucleic acid molecule isolated and cloned,as per U.S. patent application Ser. No. 08/725,182, filed Oct. 3, 1996,incorporated by reference herein.

[0011] The relationship between some of the tumor associated genes and atriad of genes, known as the SSX genes, is under investigation. SeeSahin, et al., supra; Tureci, et al., Cancer Res 56:4766-4772 (1996).One of these SSX genes, referred to as SSX2, was identified, at first,as one of two genes involved in a chromosomal translocation event (t(X;18)(p11.2; q 11.2)), which is present in 70% of synovial sarcomas. SeeClark, et al., Nature Genetics 7:502-508 (1994); Crew et al., EMBO J14:2333-2340 (1995). It was later found to be expressed in a number oftumor cells, and is now considered to be a tumor associated antigenreferred to as HOM-MEL-40 by Tureci, et al, supra. Its expression todate has been observed in cancer cells, and normal testes only. Thusparallels other members of the “CT” family of tumor antigens, since theyare expressed only in cancer and testis cells. Crew et al. also isolatedand cloned the SSX1 gene, which has 89% nucleotide sequence homologywith SSX2. Sequence information for SSX1 and SSX2 is presented as SEQ IDNOS: 1 and 2 respectively. See Crew et al., supra. Additional workdirected to the identification of SSX genes has resulted in theidentification of SSX3, as is described by DeLeeuw, et al., Cytogenet.Genet 73:179-183 (1996). The fact that SSX presentation parallels other,CT antigens suggested to the inventors that other SSX genes might beisolated. The parent application, supra discloses this work, as doesGure, et al. Int. J. Cancer 72:965-971 (1997), incorporated byreference.

[0012] With respect to additional literature on the SSX family, most ofit relates to SSX1. See PCT Application W/96 02641A2 to Cooper, et al,detailing work on the determination of synovial sarcoma viadetermination of SSX1 or SSX2. Also note DeLeeuw, et al. Hum. Mol. Genet4(6):1097-1099 (1995). also describing synovial sarcoma and SYT-SSX1 orSSX2 translocation. Also see Kawai, et al, N. Engl. J. Med338(3):153-160 (1998); Noguchi, et al. Int. J. Cancer 72(6):995-1002(1997), Hibshoosh, et al., Semin. Oncol 24(5):515-525 (1997), Shipley,et al., Am. J. Pathol. 148(2):559-567 (1996); Fligman, et al. Am. J.Pathol. 147(6); 1592-1599 (1995). Also see Chand, et al., Genomics30(3):545-552 (1995), Brett, et al., Hum. Mol Genet 6(9): 1559-1564(1997), deBruyn, et al, Oncogene (1313):643-648. The SSX3 gene isdescribed by deLeeuw, et al, Cytogenet Cell Genet 73(3):179-1983 (1966).

[0013] Application of a modification of the SEREX technology describedsupra has been used, together with other techniques, to clone two,additional SSX genes, referred to as SSX4 and SSX5 hereafter as well asan alternate splice variant of the SSX4 gene. Specifically, while theSEREX methodology utilizes autologous serum, the methods set forth infrause allogenic serum.

[0014] Motif analysis is a tool which permits one to ascertain whatregions of a longer protein may in fact be of particular interest asbinders of MHC or HLA molecules. Essentially, one works with an aminoacid motif, which generally includes at least two, and sometimes more,defined amino acids in a sequence of 8-12 amino acids. This motif isthen used to screen a longer sequence to determine which sequenceswithin the longer sequence constitute peptides which would bind to anHLA or MHC molecule, and possibly stimulate proliferation of cytolytic Tlymphocytes with specificity to complexes of the peptide and MHC/HLAmolecule. Motifs differ for different MHC/HLA molecules. Much work hasbeen done in this area, but it is ongoing. As will be seen in thedisclosure which follows, the inventors have used motif analysis toidentify peptides which bind to HLA molecules, HLA-A2 molecules inparticular.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 depicts a peptide titration experiment showing recognitionby the CTL of COS-A201 cells pulsed with increasing amounts of the SEQID NO: 19. The four lines represent separate assays using 4 differentCTL lines derived from a stimulation culture.

[0016]FIG. 2 depicts the results of cytotoxicity assays to determine HLAspecificity of SEQ ID NO: 19.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] One embodiment of the invention is a novel immunogenic peptide,SEQ ID NO: 19 (Arg-Leu-Leu-Glu-Phe-Tyr-Leu-Ala-Met), that is capable ofstimulating proliferation of cytolytic T cells and inducing a cellularimmune response against cells with SEQ ID NO: 19 complex on the cellsurface.

[0018] SEQ ID NO: 19 may be used as a vaccine either therapeutically orprophylactically. It may be administered, for example, to a patientsuffering from a disorder to stimulate one or more components of thepatient's immune system, such as cytotoxic T lymphocytes, to mount acellular immune response against the cells presenting the complex. Thedisorder may be a neoplasia, such as a melanoma. SEQ ID NO: 19 may beadministered directly. Preferably, SEQ ID NO: 19 is administered as acomplex with an HLA molecule or an HLA molecule fragment to stimulateHLA specific cytotoxic lymphocyte reaction.

[0019] In prophylactic usage, SEQ ID NO: 19 or SEQ ID NO: 19 conjugatedto a HLA molecule is provided to a patient who is in a high risk groupfor developing a disorder such as a neoplastic disorder. High riskgroups may include patients with a family history of neoplasticdisorders, patients with a genetic predisposition to certain neoplasticdisorders, and patients with habits and lifestyles which predispose themto a high risk of such disorders. Examples of high risk groups include,for example, Xeroderma Pigmentosum patients who have an increased riskfor melanoma, cigarette smokers who have an increased risk for smallcell lung carcinoma, and Beckwith-Wiedemann syndrome patients who havean increased risk for hepatoblastoma, and adrenal carcinoma. Inprophylactic usage, SEQ ID NO: 19 by itself or complexed with an HLAmolecule is administered in advance of any indications of neoplasticdisorder to stimulate the patient's immune system to prevent neoplasticdisorder or to attenuate any undiscovered neoplastic disorder.Prophylactic usage also includes the administration of SEQ ID NO: 19 toa patient recovering from a neoplastic disorder treatment such as a bonemarrow transplant or tumor excision to prevent re-emergence of theneoplasia.

[0020] In a preferred embodiment, mammals, including humans, who are athigh risk for a neoplastic disorder are treated with vaccines comprisingSEQ ID NO: 19 or SEQ ID NO: 19 complexed with HLA. The vaccine may be inthe form of a virus which can infect a cell and induce presentation ofSEQ ID NO: 19 on the cell surface. Alternatively, the vaccine may be anattenuated bacterium, which expresses SEQ ID NO: 19 and a HLA moleculeon its cell surface. In one embodiment, the vaccine may be a mammaliancell transfected by a nucleic acid molecule that encodes SEQ ID NO: 19.

[0021] Another embodiment of the invention is directed to the use of SEQID NO: 19 to generate antisera for prophylactic or treatment purposes.Antisera may be produced using methods known in the art. For example,SEQ ID NO: 19 by itself or in complex with HLA molecules may be injectedinto animals. Antibody titer to SEQ ID NO: 19 may be monitored bywithdrawing blood from the animals at regular intervals and analyzingserum titer. Booster shots of additional SEQ ID NO: 19 or SEQ ID NO:19/HLA complexes to stimulate antibody production may be administered ifneeded. Alternatively, a blood sample from a patient treated with SEQ IDNO: 19 either therapeutically or prophylactically may be used to prepareantisera.

[0022] In addition, monoclonal antibodies to SEQ ID NO: 19 may be madeusing conventional monoclonal antibody techniques. Further, to improvethe efficacy of the monoclonal antibody, it may be humanized. In analternative embodiment, antibodies or antisera may be made in a nonhumanmammal and used. It is understood that all antibodies and antisera maybe purified by antigen immunoaffinity column. In antigen immunoaffinitypurification, the antigen which is immobilized on the solid phase may beSEQ ID NO: 19 alone or SEQ ID NO: 19 complexed with a HLA molecule orHLA molecule fragment. Preferred HLA molecules include HLA-A2 moleculessuch as HLA-A*0201.

[0023] The antibodies and antisera produced may be conjugated to toxicmolecules or a detectable label and used to target neoplastic cells fordestruction or detection. A toxin may include, for example, ricin.Detectable labels may include, for example, radioisotopes ³²P, ¹²⁵I, and¹¹¹In. It is understood that some molecules, such as ¹³¹I, can be both adetectable label and a toxin.

[0024] Another embodiment of the invention is directed to a method ofdiagnosing a neoplastic disorder using an antibody that specificallybinds SEQ ID NO: 19. For example, a SEQ ID NO: 19 specific antibody maybe coupled to a detectable label and used in immunocytochemistry todetermine the presence of neoplastic disorder in a biopsy, in an in-situhybridization, and in fluorescent activator sorting.

[0025] In another embodiment of the invention, the antibody to SEQ IDNO: 19 may be used to purify SEQ ID NO: 19 by immunoaffinitychromatography. Protocols for antibody purification and antibodyaffinity purification are commonly known. Chromatography media forantibody purification and affinity purification are availablecommercially (e.g., Pharmacia Biotech, Uppsala, Sweden) and detailedprotocols for performing these techniques are supplied with the media.

[0026] In another embodiment of the invention, the antibody to SEQ IDNO: 19 may be used as a fluorescent activated cell sorter marker duringbone marrow sorting. For example, in an autologous bone marrowtransplant as a treatment for cancer, a patient's extracted bone marrowcells are sorted to separate the neoplastic cells from normal cells. Theneoplastic cells are discarded while the normal cells are reintroducedinto a patient after the patient has undergone cancer treatment. Thesorting (or multiple sortings) is performed with a fluorescent activatedcell sorter after the cells are contacted with fluorescent labeledneoplastic cell specific antibody. The antibodies and antisera of theinvention, specific for SEQ ID NO: 19, may be fluorescent labeled andused as the fluorescent antibody for cell sorting.

[0027] Another embodiment of the invention is directed to a method ofenhancing stem cell transplantation using SEQ ID NO: 19. Stem celltransplantation is used in the treatment of neoplastic disorder wherethe treatment (i.e., chemotherapy) destroys the stem cells of a patient.Without a stem cell transplant, the patient may die. In a stem celltransplantation, stem cells (marrow cells) are isolated from ahistocompatible donor and injected into a host. The transplanted stemcells, obtained from the marrow, generally contain a fair portion of Tlymphocytes. The T lymphocytes do not affect the stem cell transplantand are not removed prior to transplantation. Thus, T lymphocytes are apart of most stem cell transplants. If the T lymphocytes are properlystimulated, they may initiate an immune response against any possibleresidual neoplastic cells in the host. In the method, the donor stemcell population comprising T lymphocytes is treated with SEQ ID NO: 19or SEQ ID NO: 19 complexed to an HLA peptide before it is deposited intothe patient. The treatment may stimulate the immune response of the Tlymphocyte in the donor stem cell.

[0028] Another embodiment of the invention is a novel, substantiallypurified and isolated nucleic acid molecule encoding a peptide with anamino acid sequence of SEQ ID NO: 19(Arg-Leu-Leu-Glu-Phe-Tyr-Leu-Ala-Met). The nucleic acid that encodes SEQID NO: 19 may be deduced from the amino acid sequence of SEQ ID NO: 19.It is known that the nucleic acid code is degenerate. In this case, forexample, Arginine is encoded by the codons, CGT, CGC, CGA, CGG, AGA andAGG. Leucine is encoded by the codons TTA, TTG, CTT, CTC, CTA and CTG.Glutamic Acid is encoded by the codons GAA or GAG. Phe is encoded by thecodons TTT and TTC. Tyr is encoded by the codons TAT and TAC. Alanine isencoded by the codons GCT, GCC, GCA, and GCG. Met is encoded by thecodon ATG. Thus, the nucleic acid molecule may have any sequence whichencodes a peptide of SEQ ID NO: 19. Preferably, the nucleic acid is inoperable linkage with a promoter which can express SEQ ID NO: 19constitutively or upon induction in a eukaryotic or prokaryotic host.More preferably, the plasmid may have a wide host range allowingreplication multiple hosts such as yeast and bacteria.

[0029] Another embodiment of the invention is directed to novelpharmaceutical compositions useful for treating a neoplastic disorder.The pharmaceutical composition contains between 0.001% to 100% by weightof SEQ ID NO: 19 in a pharmaceutically acceptable carrier and/ordiluent. Suitable carriers may be bovine serum albumin and suitablediluents may be phosphate buffered saline or distilled water. In apreferred embodiment, SEQ ID NO: 19 is bound to an HLA molecule forstimulation of the recipient's immune response. Preferred HLA moleculesinclude HLA-A2 molecules such as HLA-A*0201.

[0030] Another embodiment of the invention is a method for amelioratingthe symptoms of a neoplastic disorder by administering a pharmaceuticalcomposition comprising SEQ ID NO: 19 to a patient. Administration may beperformed by means known in the art including topical administration,injection, intravenous drip, implantation of a slow release device,topical administration and aerosol administration. The therapeuticeffects of SEQ ID NO: 19 may be enhanced by the addition of an adjuvant,a T cell booster, or a cytokine. Adjuvants and cytokines that canenhance T lymphocyte response to antigens are known and include, forexample, microorganisms such as BCG, T cell boosters such as lentinan,and cytokines such as IL-1, IL-2, IL-4, IL-6, IL-12 and TNF.

[0031] Another embodiment of the invention is directed to a host cell orvirus containing a DNA molecule which encodes the peptide of theinvention. The host cell or virus may be used in a method to produce thepeptide. Further, the host cell or virus may be used as a live, orattenuated vaccine to vaccinate against a neoplastic disorder.

[0032] Another embodiment of the invention is directed to a method toprovide a vaccine for preventing a neoplastic disorder. The vaccine maybe a virus or bacterium containing a DNA molecule that encodes a peptideof the invention.

[0033] Another embodiment of the invention is directed to a method ofmaking a peptide of the invention by culturing a virus or bacteriacontaining a DNA molecule that encodes a peptide of the invention.Methods for peptide expression are known. For example, the peptide maybe produced by transfecting an expression vector containing a nucleicacid sequence that encodes SEQ ID NO: 19 into a host cell and inducingexpression. The host cell may be a prokaryotic (bacterium), yeast,insect, or mammalian cell. Various methods of host cell expression arewell known. Reagents, vectors, cell lines and detailed expressionprotocols are commercially available (e.g., Invitrogen (Carlsbad,Calif.), Stratagene (La Jolla, Calif.)). For example, SEQ ID NO: 19 maybe expressed as a thioredoxin-SEQ ID NO: 19 fusion protein under theThioFusion™ Expression system of Invitrogen. Following expression, thefusion peptide may be purified by a metal binding resin with specificaffinity for thioredoxin part of the fusion protein. The fusion protein,still bound to the metal binding resin, may be cleaved by enterokinaseto specifically release intact SEQ ID NO: 19. Alternatively, SEQ ID NO:19 may be synthesized and purified by commercially available peptidesynthesis machines (e.g., PE Corporation (Norwalk, Conn.); AdvancedChemTech (Louisville, Ky.)).

[0034] Another embodiment of the invention is directed to a polytopemolecule comprising a plurality of sequences corresponding to amino acidsequences which bind to MHC molecules, at least one of which is theamino acid sequence of SEQ ID NO: 19. A polytope is a polypeptidecomprising two or more MHC binding sequence. A nucleic acid moleculeencoding a polytope may be made by ligating together oligonucleotidesencoding different peptides. The polytope encoding nucleic acid moleculemay be placed in an expression vector in functional linkage with apromoter for expressing a recombinant polytope in a host cell. Therecombinant polytope polypeptide of the invention may comprise from 2 to1000, preferably 4 to 200 and most preferably between 4 and 20 peptides;wherein at least one of them is SEQ ID NO: 19. In addition, the peptidesmay be sandwiched between recognition sites for a sequence specificcleavage enzyme. Sequence specific cleavage enzymes may be, for example,enterokinase or thrombin which recognizes and cleaves at knownsequences. Thus, cleavage of the polytope will result in multiplepeptides which can bind to a MHC molecule.

[0035] Another embodiment of the invention is directed to a method oftreating a patient with neoplastic disorder with SEQ ID NO: 19. In themethod, T lymphocytes isolated from a patient are treated with SEQ IDNO: 19 to sensitize the T cells to SEQ ID NO: 19. The treated Tlymphocytes are reintroduced into the patient to stimulate the patient'sresponse to neoplastic cells. Optionally, the treated T lymphocytes maybe cultured and amplified before reintroduction to enhance the effectsof SEQ ID NO: 19.

[0036] Another embodiment of the invention is directed to a compositionof matter comprising a peptide of SEQ ID NO: 19. The composition ofmatter may include, in addition to SEQ ID NO: 19, one or more disorderassociated antigens, HLA molecules or HLA molecule fragments. Thecompositions may be tailored to elicit a CTL reaction against specificcells or cell populations when the composition is administered to apatient. For example, the composition may comprise multiple cell surfacemarkers expressed by a targeted cell population. The cell surfacemarkers may include, for example, HLA antigens and disorder associatedantigens.

EXAMPLE 1

[0037] A human testicular cDNA expression library was obtained, andscreened, with serum from a melanoma patient identified as MZ2. Seee.g., parent application U.S. patent application Ser. No. 08,479,328incorporated by reference; also see U.S. patent application Ser. No.08/725,182 also incorporated by reference; Sahin, et al., Proc. Natl.Acad. Sci. USA 92:11810-11813 (1995). This serum had been treated usingthe methodology described in these references. Briefly, serum wasdiluted 1:10, and then preabsorbed with transfected E. coli lysate.Following this preabsorption step, the absorbed serum was diluted 11:10,

[0038] for a final dilution of 1:100. Following the final dilution thesamples were incubated overnight at room temperature, withnitrocellulose membranes containing phage plaques prepared using themethodology referred to supra. The nitrocellulose membranes were washed,incubated with alkaline phosphatase conjugated goat anti-humanFc_(γ)secondary antibodies, and the reaction was observed with thesubstrates 5-bromo-4-chloro-3-indolyl phosphate and nitrobluetetrazolium. In a secondary screen, any phagemids which encoded humanimmunoglobulin were eliminated.

[0039] A total of 3.6×10⁵ pfus were screened, resulting in eightpositive clones. Standard sequencing reactions were carried out, and thesequences were compared to sequence banks of known sequences.

[0040] Of the eight clones, two were found to code for known autoimmunedisease associated molecules, i.e., Golgin-95 (Fritzler, et al., J. Exp.Med.178:49-62 (1993)), and human upstream binding factor (Chan, et al.,J. Exp. Med. 174:1239-1244 (1991)). Three other clones were found toencode for proteins which are widely expressed in human tissue, i.e.,ribosomal receptor, collagen type VI globular domain, and rapamycinbinding protein. Of the remaining three sequences, one was found to benon-homologous to any known sequence, but was expressed ubiquitously inhuman tissues (this was found via RT-PCR analysis, but details are notprovided herein). The remaining two were found to be identical to fulllength HOM-MEL-40, described in Ser. No. 08/479,328, while the eighthclone was found to be almost identical to “SSX3,” as described byDeLeeuw, et al., Cytogenet. Cell Genet 73:179-183 (1996), differingtherefrom in only two base pair differences in the coding region. Thesedifferences are probably artifactual in nature; however, the clone alsoincluded a 43 base pair 3′-untranslated region.

EXAMPLE 2

[0041] In order to carry out Southern blotting experiments, describedinfra, the SSX genes were amplified, using RT-PCR.

[0042] To do this, two primers were prepared using the published SSX2sequence i.e., MEL-40A: 5′-CACACAGGAT CCATGAACGG AGA, (SEQ ID NO: 3)

[0043] and

[0044] MEL-40B: (SEQ. ID NO: 4) 5′-CACACAAAGC TTTGAGGGGA GTTACTCGTC ATC

[0045] See Crew, et al., EMBO J 14:2333-2340 (1995). Amplification wasthen carried out using 0.25 U Taq polymerase in a 25 μl reaction volume,using an annealing temperature of 60° C. A total of 35 cycles werecarried out.

EXAMPLE 3

[0046] The RT-PCR methodology described supra was carried out ontesticular total RNA, and the amplification product was used in southernblotting experiments.

[0047] Genomic DNA was extracted from non-neoplastic tissue samples, andthen subjected to restriction enzyme digestion, using BamHI, Eco RI, orHindIII in separate experiments and then separated on a 0.7% agarosegel, followed by blotting onto nitrocellulose filters. The amplificationproducts described supra were labeled with ³²P, using well-knownmethods, and the labeled materials were then used as probes under highstringency conditions (65° C., aqueous buffer), followed by highstringency washes, ending with a final wash at 0.2×SSC, 0.2% SDS, 65° C.

[0048] The Southern blotting revealed more than 10 bands, in each case(i.e., each of the BamHI, EcoRI, and HindIII digests), stronglysuggesting that there is a family of SSX genes which contained more thanthe three identified previously. In view of this observation, anapproach was designed which combined both PCR cloning, and restrictionmap analysis, to identify other SSX genes.

EXAMPLE 4

[0049] When the sequences of SSX1, 2 and 3 were compared, it was foundthat they shared highly conserved 5′ and 3′ regions, which explained whythe olignucleotides of SEQ ID NOS: 3 and 4 were capable of amplifyingall three sequences under the recited conditions, and suggested thatthis homology was shared by the family of SSX genes, whatever its size.Hence, the oligonucleotides of SEQ ID NOS: 3 and 4 would be sufficientto amplify the other members of the SSX gene family.

[0050] An analysis of the sequences of SSX1, 2 and 3 revealed that SSX1and 2 contained a BglII site which was not shared by SSX3. Similarly,SSX3 contained an EcoRV site not shared by the other genes.

[0051] In view of this information, testicular cDNA was amplified, usingSEQ ID NOS: 3 and 4, as described supra, and was then subjected to BglIIdigestion. Any BglII resistant sequences were then cloned, sequenced,and compared with the known sequences.

[0052] This resulted in the identification of two previouslyunidentified sequences, referred to hereafter as SSX4 and SSX5,presented as SEQ ID NOS: 5 and 6 herein. A search of the GenBankdatabase found two clones, identified by Accession Number N24445 andWO0507, both of which consisted of a sequence—tag—derived cDNA segment.The clone identified by N24445 contained the 3′-untranslated region ofSSX4, and part of its coding sequence, while the one identified asWO0507 contained a shorter fragment of the 3′-untranslated region ofSSX4, and a longer part of the coding sequence. Specifically, N24445consists of base 344 of SSX4 (SEQ ID NO:5), through the 3-end, plus 319bases 3′ of the stop codon. The W00507 sequence consists of a 99 basepair sequence, showing no homology to SSX genes followed by a regionidentical to nucleotides 280 through the end of SEQ ID NO:5, through 67bases 3′ of the stop codon of SEQ ID NO: 1.

[0053] Two forms of SSX4 (SEQ ID NO: 5) were identified. One of theselacked nucleotides 331 to 466 but was otherwise identical to SSX4 aspresented in SEQ ID NO: 5. As is described infra, the shorter form is analternatively spliced variant.

[0054] In Table 1, which follows, the nucleotide and amino acidsequences of the 5 known members of the SSX family are compared. Onereads the table horizontally for nucleotide homology, and vertically foramino acid homology. TABLE 1 Nucleotide and amino acid homology amongSSX family members Nucleotide Sequence Homology (%) SSX1 SSX2 SSX3 SSX4SSX5 SSX1 89.1 89.6 89.4 88.7 SSX2 78.2 95.1 91.5 92.9 SSX3 77.7 91.091.1 92.7 SSX4 79.3 79.8 80.9 89.8 SSX5 76.6 83.5 84.0 77.7 Amino AcidSequence Homology (%)

[0055] Hence, SSX1 and SSX4 share 89.4% homology on the nucleotidelevel, and 79.3% homology on the amino acid level.

[0056] When the truncated form of SSX4 is analyzed, it has an amino acidsequence completely different from others, due to alternate splicing andshifting of a downstream open reading frame. The putative protein is 153amino acids long, and the 42 carboxy terminal amino acids show nohomology to the other SSX proteins.

EXAMPLE 5

[0057] The genomic organization of the SSX2 genes was then studied. Todo this, a genomic human placental library (in lambda phage) wasscreened, using the same protocol and probes described supra in thediscussion of the southern blotting work. Any positive primary cloneswere purified, via two additional rounds of cloning.

[0058] Multiple positive clones were isolated, one of which waspartially sequenced, and identified as the genomic clone of SSX2. Aseries of experiments carrying out standard subcloning and sequencingwork followed, so as to define the exon-intron boundaries.

[0059] The analysis revealed that the SSX2, gene contains six exons, andspans at least 8 kilobases. All defined boundaries were found to observethe consensus sequence of exon/intron junctions, i.e. GT/AG.

[0060] The alternate splice variant of SSX4, discussed supra, was foundto lack the fifth exon in the coding region. This was ascertained bycomparing it to the SSX2 genomic clone, and drawing correlationstherefrom.

EXAMPLE 6

[0061] The expression of individual SSX genes in normal and tumortissues was then examined. This required the construction of specificprimers, based upon the known sequences, and these follow, as SEQ IDNOS: 7-16: TABLE 2 Gene-specific PCR primer sequences for individual SSXgenes SSX 1A (5′): 5′-CTAAAGCATCAGAGAAGAGAAGC [nt.44-66] SSX 1B (3′):5′-AGATCTCTTATTAATCTTCTCAGAAA [nt.440-65] SSX 2A (5′):5′-GTGCTCAAATACCAGAGAAGATC [nt.41-63] SSX 2B (3′):5′-TTTTGGGTCCAGATCTCTCGTG [nt.102-25] SSX 3A (5′):5′-GGAAGAGTGGGAAAAGATGAAAGT [nt.454-75] SSX 3B (3′):5′-CCCCTTTTGGGTCCAGATATCA [nt.458-79] SSX 4A (5′):5′-AAATCGTCTATGTGTATATGAAGCT [nt.133-58] SSX 4B (3′):5′-GGGTCGCTGATCTCTTCATAAAC [nt.526-48] SSX 5A (5′):5′-GTTCTCAAATACCACAGAAGATG [nt.39-63] SSX 5B (3′):5′-CTCTGCTGGCTTCTCGGGCCG [nt.335-54]

[0062] The specificity of the clones was confirmed by amplifying thepreviously identified cDNA for SSX1 through SSX5. Taq polymerase wasused, at 60° C. for SSX1 and 4, and 65° C. for SSX2, 3 and 5. Each setof primer pairs was found to be specific, except that the SSX2 primerswere found to amplify minute (less than {fraction (1/20)} of SSX2)amounts of SSX3 plasmid DNA.

[0063] Once the specificity was confirmed, the primers were used toanalyze testicular mRNA, using the RT-PCR protocols se t forth supra.

[0064] The expected PCR products were found in all 5 cases, andamplification with the SSX4 pair did result in two amplificationproducts, which is consistent with alternative splice variants.

[0065] The expression of SSX genes in cultured melanocytes was thenstudied. RT-PCR was carried out, using the protocols set forth supra. NoPCR product was found. Reamplification resulted in a small amount ofSSX4 product, including both alternate forms, indicating that SSX4expression in cultured melanocytes is inconsistent and is at very lowlevels when it occurs.

[0066] This analysis was then extended to a panel of twelve melanomacell lines. These results are set forth in the following table. TABLE 3SSX expression in melanoma cell lines detected by RT-PCR* SSX1 SSX2 SSX3SSX4 SSX5 MZ2-Mel 2.2 + + − − − MZ2-Mel 3.1 + + − − − SK-MEL-13 − − − −− SK-MEL-19 − − − − − SK-MEL-23 − − − − − SK-MEL-29 − − − − − SK-MEL-30 −*  −* −  −* − SK-MEL-31 − − − − − SK-MEL-33 − − − − − SK-MEL-37 + +− + + SK-MEL-179 − − − − − M24-MET − − − − −

EXAMPLE 7

[0067] Additional experiments were carried out to analyze expression ofthe members of the SSX family in various tumors. To do this, totalcellular RNA was extracted from frozen tissue specimens using guanidiumisothiocyanate for denaturation followed by acidic phenol extraction andisopropanol precipitation, as described by Chomczynski, et al, Ann.Biochem 162: 156-159 (1987), incorporated by reference. Samples of totalRNA (4 ug) were primed with oligoDT(18) primers, and reversetranscribed, following standard methodologies. The integrity of the cDNAthus obtained was tested via amplifying B-acin transcripts in a 25cycle, standard PCR, as described by Tureci, et al, Canc. Res. 56:4766-4772 (1996).

[0068] In order to carry out PCR analyses, the primers listed as SEQ IDNOS: 5-14, supra were used, as well as SEQ ID NOS: 17 and 18, i.e.:ACAGCATTAC CAAGGACAGC AGCCACC GCCAACAGCA AGATOCATAC CAGGGAC

[0069] These two sequences were each used with both SEQ ID NOS: 6 and 8in order to detect the SYT/SSX fusion transcript reported for synovialsarcoma by Clark et al, supra, and Crew, et al, supra. The amplificationwas carried out by amplifying 1 μl of first strand cDNA with 10 pMol ofeach dNTP, and 1.67 mN MgCl₂ in a 30 μl reaction. Following 12 minutesat 94° C. to activate the enzyme, 35 cycles of PCR were performed. Eachcycle consisted of 1 minute for annealing (56° C. for SEQ ID NOS: 7 & 8;67° C. for SEQ ID NOS: 9 & 10; 65° C. for SEQ ID NOS: 11& 12; 60° C. forSEQ ID NOS: 13 & 14; 66° C. for SEQ ID NOS: 15 & 16; 60° C. for SEQ IDNOS: 17 & 8 and 18 & 10), followed by 2 minutes at 72° C., 1 minute at94° C., and a final elongation step at 72° C. for 8 minutes. A 15 μlaliquot of each reaction was size fractionated on a 2% agarose gel,visualized with ethidium bromide staining, and assessed for expectedsize. The expected sizes were 421 base pairs for SEQ ID NOS: 7 & 8; 435base pairs for SEQ ID NOS: 9 & 10; 381 base pairs for SEQ ID NOS 11 &12; 413 base pairs for SEQ ID NOS: 13 & 14, and 324 base pairs for SEQID NOS: 15 & 16. The conditions chosen were stringent, so as to preventcross anneling of primers to other members of the SSX family. Additionalsteps were also taken to ensure that the RT-PCR products were derivedfrom cDNA, and not contaminating DNA. Each experiment was done intriplicate. A total of 325 tumor specimens were analyzed. The resultsare presented in Tables 4 & 5 which follow.

[0070] It is to be noted that while most of the SSX positive tumorsexpressed only one member of the SSX family, several tumor types showedcoexpression of two or more genes.

[0071] Expression of SSX genes in synovial sarcoma was analyzed, becausethe literature reports that all synovial sarcoma cases analyzed havebeen shown to carry either the SYT/SSX1 or SYT/SSX2 translocation, atbreakpoints flanked by the primer sets discussed herein, i.e., SEQ IDNO: 17/SEQ ID NO: 8; SEQ ID NO: 17/SEQ ID NO: 10; SEQ ID NO. 17/SEQ IDNO: 8; SEQ ID NO: 18/SEQ ID NO: 10. The PCR work described supra showedthat SYT/SSX1 translocations were found in three of the synovial sarcomasamples tested, while SYT/SSX2 was found in one. The one in which it wasfound was also one in which SYT/SSX1 was found. Expression of SSXappeared to be independent of translocation. TABLE 4 Expression of SSXgenes by human neoplasms Tissues at lease one Tumor entity tested SSX1SSX2 SSX3 SSX4 SSX5 positive % Lymphoma 11 — 4 — — — 4 36 Breast cancer67 5 5 — 10  — 16 23 Endometrial cancer 8 1 2 — 1 1 1 13 Colorectalcancer 58 3 7 — 9 1 16 27 Ovarian cancer 12 — — — 6 — 6 50 Renal cellcancer 22 — 1 — — — 1 4 Malignant melanoma 37 10  13  — 10  2 16 43Glioma 31 — 2 — 3 — 5 16 Lung cancer 24 1 4 — 1 1 5 21 Stomach cancer 3— — — 1 — 1 33 Prostatic cancer 5 — 2 — — — 2 40 Bladder cancer 9 2 4 —2 — 5 55 Head-Neck cancer 14 3 5 — 4 1 8 57 Synovial sarcoma 4 — 2 — 1 13 75 Leukemia 23 — — — — — 0 0 Leiomyosarcoma 6 — — — — — 0 0 Thyroidcancer 4 — — — — — 0 0 Seminoma 2 — — — — — 0 0 Total 325 25  50  0 48 7 89

[0072] TABLE 5 Expression pattern of individual SSX genes inSSX-positive tumor samples.^(J) SSX1 SSX2 SSX4 SSX5 Breast Cancer (67specimens) 51 specimens − − − − 7 specimens − − + − 4 specimens − + − −2 specimens + − − − 2 specimens + − + − 1 specimen + + + − Melanoma (37specimens) 21 specimens − − − − 5 specimens + + + − 4 specimens − + − −2 specimens − + + − 1 specimen + − − − 1 specimen + + − − 1 specimen +− + − 1 specimen + − + + 1 specimen + + + + Endomet. Cancer (8specimens) 7 specimens − − − − 1 specimen + + + + Glioma (31 specimens)25 specimens − − − − 3 specimens − + − − 2 specimens − − + − Lung Cancer(24 specimens) 19 specimens − − − − 3 specimens − + − − 1 specimen − −− + 1 specimen + + + − Colorectal Cancer (58 specimens) 42 specimens − −− − 7 specimens − + − − 5 specimens − − + − 3 specimens + − + − 1specimen − − + + Bladder Cancer (9 specimens) 4 specimens − − − − 2specimens − + − − 1 specimen − − + − 1 specimen + + − − 1 specimen + + +− Head-Neck Cancer (14 specimens) 6 specimens − − − − 2 specimens + − −− 2 specimens − + + − 1 specimen − + − − 1 specimen − − + − 1specimen + + − − 1 specimen − + + + SSX1 SSX2 SSX4 SSX5 SYT/SSX1SYT/SSX5 Synovial Sarcoma (4 specimens) Sy1 − − + − + − Sy2 − + − + + −Sy3 − − − − − + Sy4 − + − − + −

EXAMPLE 8

[0073] This example details further experiments designed to identifyadditional peptides which bind to HLA-A2 molecules, and which stimulateCTL proliferation.

[0074] First, peripheral blood mononuclear cells (“PBMCs” hereafter)were isolated from the blood of healthy HLA-A*0201⁺ donors, usingstandard Ficoll-Hypaque methods. These PBMCs were then treated toseparate adherent monocytes from non-adherent peripheral bloodlymphocytes (“PBLs”), by incubating the cells for 1-2 hours, at 37° C.,on plastic surfaces. Any non-adherent PBLs were cryopreserved untilneeded in further experiments. The adherent cells were stimulated todifferentiate into dendritic cells by incubating them in AIMV mediumsupplemented with 1000 U/ml of IL-4, and 1000 U/ml of GM-CSF. The cellswere incubated for 5 days.

[0075] Seven days after incubation began, samples of the dendritic cells(8×10⁵) were loaded with 50 μg/ml of exogenously added peptide. (Detailsof the peptides are provided infra). Loading continued for 2 hours, at37° C., in a medium which contained 1000 U/ml of TNF-α, and 10,000 U/mlIL-1β. The peptide pulsed dendritic cells were then washed, twice, inexcess, peptide free medium. Autologous PBLs, obtained as described,supra, were thawed, and 4×10⁷ PBLs were then combined with 8×10⁵ peptideleaded dendritic cells, (ratio: 50:1), in a medium which contained 5ng/ml of IL-7 and 20 U/ml of IL-2. The cultures were then incubated at37° C.

[0076] Lymphocyte cultures were restimulated at 14, 21, and 28 days, inthe same manner as the experiment carried out after 7 days. Cytotoxicityassays were carried out, at 14, 21, and 28 days, using a europiumrelease assay, as described by Blomberg, et al., J. Immunol. Meth. 114:191-195 (1988), incorporated by reference, or the commercially availableELISPOT assay, which measures IFN-γ release.

[0077] The peptides which were tested were all derived from the aminoacid sequence of NY-ESO-1 as is described in U.S. Pat. No. 5,804,381, toChen, et al., incorporated by reference, or the amino acid sequences ofSSX-4. The peptides tested were:

[0078] RLLEFYLAM (SEQ ID NO: 19) and

[0079] SLAQDAPPL (SEQ ID NO: 20)

[0080] both of which are derived from NY-ESO-1, and

[0081] STLEKINKT (SEQ ID NO: 21)

[0082] derived from SSX-4. The two NY-ESO-1 derived peptides were testedin ELISPOT assays. The results follow. In summary, three experimentswere carried out. The results are presented in terms of the number ofspots (positives) secured when the HLA-A2 positive cells were pulsedwith the peptide minus the number of spots obtained using non-pulsedcells. As indicated, measurements were taken at 14, 21 and 28 days.

[0083] The following results are for peptide RLLEFYLAM (SEQ ID NO. 19).Day Measured (Pulsed Cells - Unpulsed Cells) 14 21 28 Expt 1 30 8 * Expt2 22 * 12 Expt 3 6 * 12

EXAMPLE 9

[0084] In follow up experiments, the T cell cultures described suprawere tested on both COS cells which had been transfected with HLA-A*0201encoding cDNA and were pulsed with endogenous peptide, as describedsupra, or COS cells which had been transfected with both HLA-A*0201 andNY-ESO-1 encoding sequences. Again, the ELISPOT assay was used, for bothtypes of COS transfectants. Six different cultures of T cells weretested, in two experiments per culture. Pulsed with Endogenous PeptideNY-ESO-1 Production Culture 1 Expt 1 64 44 Expt 2 44 52 Culture 2 Expt 148 45 Expt 2 100 64 Culture 3 Expt 1 20 37 Expt 2 16 16 Culture 4 Expt 117 40 Expt 2 28 34 Culture 5 Expt 1 36 26 Expt 2 4 36 Culture 6 Expt 112 62 Expt 2 44 96

[0085] The fact that the endogenous NY-ESO-1 led to lysis suggests thatNY-ESO-1 is processed to this peptide via HLA-A2 positive cells.

[0086] Similar experiments were carried out with the second NY-ESO-1derived peptide, i.e., SLAQDAPPL (SEQ ID NO. 20). These results follow:Pulsed with Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 2816 Expt 2 30 14 Culture 2 Expt 1 31 75 Expt 2 30 70 Culture 3 Expt 1 3244

EXAMPLE 10

[0087] In further experiments, the specificity of the CTLs generated inthe prior experiment was tested by combining these CTLs with COS cells,transfected with HLA-A*0201 encoding sequences, which were then pulsedwith peptide. First, the peptide RLLEFYLAM (SEQ ID NO. 19) was tested,in three experiments, and then SLAQDAPPL (SEQ ID NO. 20) was tested, insix experiments. Europium release was measured, as described supra, andthe percent of target cells lysed was determined. The results follow: %LYSIS Peptide Added No Peptide PEPTIDE RLLEFYLAM (SEQ ID NO. 19) Expt 143 0 Expt 2 8 0 Expt 3 9 0 PEPTIDE SLAQDAPPL (SEQ ID NO. 20) Expt 1 11 0Expt 2 13 0 Expt 3 13 0 Expt 4 21 0 Expt 5 12 0 Expt 6 42 0

[0088] In additional experiments, the CTLs specific to RLLEFYLAM (SEQ IDNO. 19)/HLA-A2 complexes also recognized and lysed melanoma cell lineSK-Mel-37 which is known to express both HLA-A2 and NY-ESO-1. Thisrecognition was inhibited via preincubating the target cells with anHLA-A2 binding monoclonal antibody, BB7.2. This confirmed that the CTLswere HLA-A2 specific for the complexes of the peptide and HLA-A2.

[0089] In a second set of experiments, a peptide titration experimentwas performed to further determine the ability of SEQ ID NO: 19 toinduce a CTL response. Experiments were conducted substantially inaccordance with the protocol of Example 8. Samples of the cells (8×10⁵)were loaded with 1 μM, 5 μM, 10 μM and 50 μM of SEQ ID NO: 19 for 2hours at 37° C. in a medium containing 1000 U/ml of TNF-α. Cytotoxicityassays were performed using a europium release assay as described supra.In four separate sets of experiments, increasing concentrations of SEQID NO: 19 cause an increase in lysis. The data is plotted in FIG. 1. Theresults indicate that cells pulsed with SEQ ID NO: 19 can elicit a CTLmediated lysis.

[0090] To determine if the lysis is HLA specific, the CTL assays wereperformed using COS cells transfected with a cDNA encoding HLA-A*0201 asdescribed supra and pulsed with SEQ ID NO: 19. As shown in FIG. 2,transfected COS cells that were not pulsed with peptides had a lysisrate of 5%. HLA-A*0201 transfected COS cells pulsed with SEQ ID NO: 19,showed 55% lysis. This is consistent with the hypothesis that SEQ ID NO:19, when complexed with an HLA molecule, is responsible for increasedlysis. To determine if the effect was HLA restricted, HLA-A*0201transfected COS cells pulsed with SEQ ID NO: 19 were assayed in thepresence of anti-HLA-A2 antibodies. The lysis decreased to about 10%.This indicated that the cytotoxicity is HLA-A2 restricted because theanti-HLA-A2 antibody bound to the HLA-A2 molecule, interfering with therecognition of the HLA-A2/SEQ ID NO: 19 complex by the TCR (T cellreceptor) of the specific T cell, thus inhibiting lysis. Similarexperiments using the SK-MEL-37 cell line, which is known to expressboth NY-ESO-1 and HLA-A2, as the target demonstrated that the peptidewas endogenously processed and presented in tumor cells. SK-MEL-37 cellsshowed about 23% lysis whereas lysis was reduced to 6% in the presenceof anti-HLA-A2, which is consistent with the findings using COS-A201cells, described supra.

[0091] To determine the cellular location of NY-ESO-1, several celllines known to express NY-ESO-1 were stained by immunofluorescence witha NY-ESO-1 specific monoclonal antibody (Stockert et al., J. Exp Med 1871349-54, 1998; incorporated herein by reference. Also see U.S. patentapplication Ser. No. 09/062,422 filed Apr. 17, 1998, incorporated hereinby reference). Upon analysis of the stained cell samples, it was foundthat NY-ESO-1 was localized to the endoplasmic reticulum. This isconsistent with the immunogenicity of NY-ESO-1.

EXAMPLE 11

[0092] An additional peptide derived from SSX-4, i.e., STLEKINKT (SEQ IDNO: 21) was also tested, in the same way the NY-ESO-1 derived peptideswere tested. First, ELISPOT assays were carried out, using COS cellswhich expressed HLA-A*0201, and which either expressed full lengthSSX-4, due to transfection with cDNA encoding the protein, or which werepulsed with the peptide. Three cultures were tested, in two experiments.The results follow: Pulsed with Endogenous Peptide NY-ESO-1 ProductionCulture 1 Expt 1 50 100 Expt 2 20 138 Culture 2 Expt 1 8 12 Expt 2 6 14Culture 3 Expt 1 15 47 Expt 2 14 54

[0093] Further, as with the NY-ESO-1 peptides, specificity of the CTLswas confirmed, using the same assay as described supra, i.e., combiningthe CTLs generated against the complexes with COS cells, transfectedwith HLA-A*0201, and pulsed with peptide. The europium release assaydescribed supra was used. The results follow: % LYSIS Peptide Added NoPeptide Expt 1 22 0 Expt 2 14 0 Expt 3 46 0 Expt 4 16 0

[0094] As with the NY-ESO-1 derived peptides, CTL recognition wasinhibited via preincubation with the monoclonal antibody BB7.2,confirming specificity of the CTL for complexes HLA-A2 and peptides.

EXAMPLE 12

[0095] Additional experiments were carried out on peptides derived fromSSX-2 i.e, KASEKIFYV, and peptides derived from NY-ESO-1, i.e.,SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT. In each case, the same type ofassays as were carried out in examples 8-11 were carried out. Theresults were comparable, in that for each peptide, CTL were generatedwhich were specific for the respective peptide/HLA-A2 complex.

EXAMPLE 13

[0096] The amino acid sequence of the proteins encoded by the SSX geneswere analyzed for peptide sequences which correspond to HLA bindingmotifs. This was done using the algorithm taught by Parker et al., J.Immunol. 142: 163 (1994), incorporated by reference, augmented by using,as an additional motif, nonamers where position 2 is Thr or Ala, andposition 9 is Thr or Ala. In the information which follows, the aminoacid sequence, the HLA molecule to which it presumably binds, and thepositions in the relevant SSX molecule are given. The resultingcomplexes should provoke a cytolytic T cell response. This could bedetermined by one skilled in the art following methods taught by, e.g.,van der Bruggen, et al., J. Eur. J. Immunol. 24: 3038-3043 (1994),incorporated by reference, as well as the protocols set forth inExamples 8-11, supra. SSX-5 A2 KASEKIIYV 41-49 (SEQ ID NO: 22) DAFVRRPRV 5-13 (SEQ ID NO: 23) QIPQKMQKA 16-24 (SEQ ID NO: 24) MTKLGFKAT 58-66(SEQ ID NO: 25) MTFGRLQGI  99-107 (SEQ ID NO: 26) NTSEKVNKT 146-154 (SEQID NO: 27) YVYMKRKYEA 48-57 (SEQ ID NO: 28) YMKRKYEAMT 50-59 (SEQ ID NO:29) EAMTKLGFKA 56-65 (SEQ ID NO: 30) MTKLGFKATL 58-67 (SEQ ID NO: 31)RLQGIGPKIT 103-112 (SEQ ID NO: 32) QLRPSGKLNT 138-147 (SEQ ID NO: 33) A3GIFPKITPEK 106-115 (SEQ ID NO: 34) KLNTSEKVNK 144-153 (SEQ ID NO: 35)A24 KYEAMTKLGF 54-63 (SEQ ID NO:36) B7 HPQMTFGRL  96-104 (SEQ ID NO: 37)GPQNNGKQL 131-139 (SEQ ID NO: 38) B8 RVRERKQL 167-174 (SEQ ID NO: 39)B44 YEAMTKLGF 55-63 (SEQ ID NO: 40) RERKQLVIY 169-177 (SEQ ID NO: 41)B52 KQLVIYEEI 172-180 (SEQ ID NO: 42) MTFGRLQGIF  99-108 (SEQ ID NO: 43)SSX-4 A2 KSSEKIVYV 41-49 (SEQ ID NO: 44) VMTKLGFKV 57-65 (SEQ ID NO: 45)YVYMKLNYEV 48-57 (SEQ ID NO: 46) KLNYEVMTKL 52-61 (SEQ ID NO: 47)FARRPRDDA  7-13 (SEQ ID NO: 48) QISEKLRKA 16-24 (SEQ ID NO: 49)MTFGSLQRI  99-107 (SEQ ID NO: 50) SLQRIFPKI 103-111 (SEQ ID NO: 51)KJVYVYMKL 45-53 (SEQ ID NO: 52) KLRKAFDDI 20-28 (SEQ ID NO: 53)KLRKAFDDIA 20-29 (SEQ ID NO: 54) YMKLNYEVMT 50-59 (SEQ ID NO: 55)MTKLGFKVTL 58-67 (SEQ ID NO: 56) QLCPPGNPST 138-147 (SEQ ID NO: 57) A3KLNYEVMTK 52-60 (SEQ ID NO: 58) A24 NYEVMTKLGF 54-63 (SEQ ID NO:59) B7RPQMTFGSL  96-104 (SEQ ID NO: 60) KPAEEENGL 115-123 (SEQ ID NO: 61)GPQNDGKQL 131-139 (SEQ ID NO: 62) CPPGNPSTL 140-148 (SEQ ID NO: 63) B8RLRERKQL 167-174 (SEQ ID NO: 64) B35 RPRDDAQI 10-17 (SEQ ID NO: 65)KPAEEENGL 115-123 (SEQ ID NO: 66) B44 YEVMTKLGF 55-63 (SEQ ID NO: 67)RERKQLVVY 169-177 (SEQ ID NO: 68) B52 KQLVVYEEI 172-180 (SEQ ID NO:69)MTFGSLQRIF  99-108 (SEQ ID NO: 70) SSX-2 A2 KIQKAFDDI 20-28 (SEQ ID NO:71) KASEKIFYV 41-49 (SEQ ID NO: 72) AMTKLGFKA 57-65 (SEQ ID NO: 73)RLQGISPKI 103-111 (SEQ ID NO: 74) RLRERKQLV 167-175 (SEQ ID NO: 75)DAFARRPTV  5-13 (SEQ ID NO: 76) FARRPTVGA  7-15 (SEQ ID NO: 77)QIPEKIQKA 16-24 (SEQ ID NO: 78) MTFGRLQGI  99-107 (SEQ ID NO: 79)ELCPPGKPT 138-146 (SEQ ID NO: 80) YVYMKRKYEA 48-57 (SEQ ID NO: 81)EAMTKLGFKA 56-65 (SEQ ID NO: 82) MTKLGFKATL 58-67 (SEQ ID NO: 83)RAEDFQGNDL 75-84 (SEQ ID NO: 84) ELCPPGKPTT 138-147 (SEQ ID NO: 85) A3TLPPFMCNK 66-74 (SEQ ID NO:86) KIFYVYMKRK 45-54 (SEQ ID NO: 87) A24KYEAMTKLGF 54-63 (SEQ ID NO:88) B7 RPQMTFGRL  96-104 (SEQ ID NO: 89)GPQNDGKEL 131-139 (SEQ ID NO: 90) B8 RLRERKQL 167-174 (SEQ ID NO: 91)B35 FSKEEWEKM 32-40 (SEQ ID NO: 92) B44 YEAMTKLGF 55-63 (SEQ ID NO: 93)RERKQLVIY 169-177 (SEQ ID NO: 94) B52 LQGISPKIM 104-112 (SEQ ID NO: 95)KQLVIYEEI 172-180 (SEQ ID NO: 96) SSX-1 A2 AMTKLGEKV 57-65 (SEQ ID NO:97) AMTKLGFKV 56-65 (SEQ ID NO: 98) FAKRPRDDA  7-15 (SEQ ID NO: 99)KASEKRSKA 16-24 (SEQ ID NO: 100) YVYMKRNYKA 48-57 (SEQ ID NO: 101)KAMTKLGFKV 56-65 (SEQ ID NO: 102) MTKLGFKVT 58-66 (SEQ ID NO: 103)MTKLGFKVTL 58-67 (SEQ ID NO: 104) RIQVEHPQMT  91-100 (SEQ ID NO: 105)MTFGRLHRI  99-107 (SEQ ID NO: 106) A3 TLPPFMCNK 66-74 (SEQ ID NO: 107)A24 NYKAMTKLGF 54-63 (SEQ ID NO: 108) B7 HPQMTFGRL  96-104 (SEQ ID NO:109) GPQNDGKOL 131-139 (SEQ ID NO: 110) B8 RLRERKQL 167-174 (SEQ ID NO:111) B44 RERKQLVIY 169-177 (SEQ ID NO: 112) B52 KQLVIYEEI 172-180 (SEQID NO: 113) MTFGRLHRII  99-108 (SEQ ID NO: 114) NY- A2 SISSCLQQL 148-156(SEQ ID NO: 115) GTGGSTGDA  7-15 (SEQ ID NO: 116) RASGPGGGA 52-60 (SEQID NO: 117) GARGPESRL 79-87 (SEQ ID NO: 118) ATPMEAELA  97-105 (SEQ IDNO: 119) FTVSGNILT 126-134 (SEQ ID NO: 120) LTAADHRQL 137-145 (SEQ IDNO: 121) QLSLLMWJT 155-163 (SEQ ID NO: 122) LMWJTQCFL 159-167 (SEQ IDNO: 123) FATPMEAEL  96-104 (SEQ ID NO: 124) TVSGNTLTI 127-135 (SEQ IDNO: 125) ATGGRGPRGA 39-48 (SEQ ID NO: 126) GAPRGPHGGA 59-68 (SEQ ID NO:127) LARRSLAQDA 104-113 (SEQ ID NO: 128) ITQCFLPVFL 162-171 (SEQ ID NO:129)

[0097] The foregoing examples describe the isolation and cloning ofnucleic acid molecules for the SSX4, splice variant of SSX4, and SSX5genes as well as methods for determining expression of the various SSXgenes as a possible indication of cancer. As was indicated, supra, thesegenes are expressed in tumor cells, thereby enabling the skilled artisanto utilize these for, e.g., assaying for cancer. The determination ofexpression can be carried out via, e.g., determination of transcripts ofan SSX gene or genes, via nucleic acid hybridization, such as viapolymerase chain reaction. In a preferred embodiment, one determinespresence of a transcript of an SSX gene by contacting a sample with anucleic acid molecule which specifically hybridizes to the transcript.

[0098] The hybridization of the nucleic acid molecule to a target isindicative of expression of an SSX gene, and of the possibility ofcancer. Preferably, this is done with two primer molecules, as in apolymerase chain reaction. Determination of expression of more than oneSSX gene in the context by these assays also a part of the invention.For the convenience of the artisan, the nucleotide sequences of SSX 1and SSX2, which are known, are presented herein as SEQ ID NOS: 1 & 2.

[0099] Alternate assays are also a part of the invention. Members of theCT family are known to provoke antibodies in the individual whoexpresses a CT family member. Hence, one can carry out the assaysdescribed herein via, e.g., determining antibodies in a sample takenfrom a subject in question. Most preferably, the sample being analyzedis serum. Such assays can be carried out in any of the standard ways onedetermines antibodies, such as by contacting the sample with an amountof protein or proteins, and any additional reagents necessary todetermine whether or not the antibody binds. One approach involves theuse of immobilized protein, where the protein is immobilized in any ofthe standard ways known to the art, followed by contact with the sampleand then, e.g., anti-IgG, anti-Fc antibodies, and so forth. Conversely,presence of an SSX protein can also be determined, using antibodies inthe place of the proteins of the above described assays.

[0100] The correlation of SSX expression with cancer also suggestsvarious therapeutic methods and compositions useful in treatingconditions associated with abnormal SSX expression. “Abnormal SSXexpression” in this context may mean expression per se, or levels whichdiffer from those in a normal individual, i.e., they may be lower orhigher.

[0101] The invention envisions therapeutic approaches such as the use ofantisense molecules to inhibit or block expression. This antisensemolecules are oligonucleotides which hybridize to the nucleic acidmolecules and inhibit their expression. Preferably these are 17-50nucleotides in length. These antisense oligonucleotides are preferablyadministered in combination with a suitable carrier, such as a cationicliposome.

[0102] Other therapeutic approaches include the administration of SSXproteins per se, one or more antigenic peptides derived therefrom, aswell as so-called polytopic vaccines. These include a plurality ofantigenic peptides, untied together, preferably by linker sequences. Theresulting peptides may bind to either MHC-Class I or Class II molecules.These proteins, peptides, or polytopic vaccines may be administered incombination with an appropriate adjuvant. They may also be administeredin the form of genetic constructs which are designed to permitexpression of the protein, the peptide, the polytopic structures, etc.Peptides and polytopic structures can be expressed by so-called“minigenes” i.e., DNA molecules designed to express portions of theentire SSX molecule, or the various portions of the molecules, linkedtogether as described supra. One can formulate the therapeuticcompositions and approaches described herein such that one, or more thanone SSX protein, is used as the source of the compositions. In otherwords, if a whole protein approach is used, one SSX molecule may beused, or two or more may be combined in one formulation. For peptides,these can all be taken from one SSX molecule, or be combinations ofpeptides taken from more than one. The polytopic structures describedherein can also be made up of components of one, or more than one, SSXmolecule.

[0103] The amount of agent administered and the manner in which it isadministered will, vary, based on the condition being treated and theindividual. Standard forms of administration, such as intravenous,intradermal, subcutaneous, oral, rectal and transdermal administrationcan be used. With respect to formulations, the proteins and or peptidesmay be combined with adjuvant and/or carriers such as a saponin, GM-CSF,one or more interleukin, an emulsifying oil such as vitamin E, one ormore heat shock protein, etc.

[0104] When the nucleic acid approach is utilized, various vectors, suchas Vaccinia or adenovirus based vectors can be used. Any vector usefulin eukaryotic transfection, such as in transfection of human cells, canbe used. These vectors can be used to produce, e.g., cells such asdendritic cells which present relevant peptide/MHC complexes on theirsurface. The cells can then be rendered non-proliferative prior to theiradministration, using standard methodologies.

[0105] Also a part of the invention are peptides which consist of aminoacid sequences corresponding to portions of SSX molecules, or theNY-ESO-1 molecule, such as those peptide sequences described supra. Ashas been shown, such peptides bind to MHC molecules, such as HLA-A2molecules, and provoke proliferation of cytolytic T cells against theformed complexes. As it has been shown that cells which express the fulllength molecules (NY-ESO-1, or SSX molecules) are in fact recognized byCTLs which were generated following pulsing of cells with relevantpeptides. This result indicates that both the peptides and CTLs shouldbe useful therapeutic agents. Hence, an additional aspect of theinvention is the administration of one or more peptides, derived fromNY-ESO-1 or an SSX molecule as described, alone or in combination, suchas in antigen “cocktails.” Such cocktails can include a mixture ofpeptides, which have been formulated following typing of a particularpatient's HLA type. Similarly, CTLs, developed in vitro, can beadministered to the patient, in view of the recognition that thepeptides are presented following endogenous expression of the fulllength molecule.

[0106] It is to be pointed out that when an MHC molecule is mentioned,such as HLA-A2, this is meant to include all allelic forms of thatmolecule. There are various types of HLA-A2 molecules which are known,and while these differ in a few amino acids, the degree of disparity isgenerally less than 10 amino acids over the full length of the molecule,and the differences are not expected to impact the ability of the formof the molecule to bind to peptides. Hence, a peptide which binds to anHLA-A*0201 molecule may by presumed to also bind to HLA-A*0202,HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0209, and soforth.

[0107] Other aspects of the invention will be clear to the skilledartisan and need not be reiterated herein.

[0108] The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, it beingrecognized that various modifications are possible within the scope ofthe invention.

1 129 1 766 DNA Homo sapiens 1 cactttgtca ccaactgctg ccaactcgccaccactgctg ccgcaatcgc aaccactgct 60 ttgtctctga agtgagactg ctcctggtgccatgaacgga gacgacacct ttgcaaagag 120 acccagggat gatgctaaag catcagagaagagaagcaag gcctttgatg atattgccac 180 atacttctct aagaaagagt ggaaaaagatgaaatactcg gagaaaatca gctatgtgta 240 tatgaagaga aactataagg ccatgactaaactaggtttc aaagtcaccc tcccaccttt 300 catgtgtaat aaacaggcca cagacttccaggggaatgat tttgataatg accataaccg 360 caggattcag gttgaacatc ctcagatgactttcggcagg ctccacagaa tcatcccgaa 420 gatcatgccc aagaagccag cagaggacgaaaatgattcg aagggagtgt cagaagcatc 480 tggcccacaa aacgatggga aacaactgcaccccccagga aaagcaaata tttctgagaa 540 gattaataag agatctggac ccaaaagggggaaacatgcc tggacccaca gactgcgtga 600 gagaaagcag ctggtgattt atgaagagatcagtgaccct gaggaagatg acgagtaact 660 cccctggggg atacgacaca tgcccttgatgagaagcaga acgtggtgac ctttcacgaa 720 catgggcatg gctgcggctc cctcgtcatcaggtgcatag caagtg 766 2 931 DNA Homo sapiens 2 actttctctc tctttcgattcttccatact cagagtacgc acggtctgat tttctctttg 60 gattcttcca aaatcagagtcagactgctc ccggtgccat gaacggagac gacgcctttg 120 caaggagacc cacggttggtgctcaaatac cagagaagat ccaaaaggcc ttcgatgata 180 ttgccaaata cttctctaaggaagagtggg aaaagatgaa agcctcggag aaaatcttct 240 atgtgtatat gaagagaaagtatgaggcta tgactaaact aggtttcaag gccaccctcc 300 cacctttcat gtgtaataaacgggccgaag acttccaggg gaatgatttg gataatgacc 360 ctaaccgtgg gaatcaggttgaacgtcctc agatgacttt cggcaggctc cagggaatct 420 ccccgaagat catgcccaagaagccagcag aggaaggaaa tgattcggag gaagtgccag 480 aagcatctgg cccacaaaatgatgggaaag agctgtgccc cccgggaaaa ccaactacct 540 ctgagaagat tcacgagagatctggaccca aaagggggga acatgcctgg acccacagac 600 tgcgtgagag aaaacagctggtgatttatg aagagatcag cgaccctgag gaagatgacg 660 agtaactccc ctcagggatacgacacatgc ccatgatgag aagcagaacg tggtgacctt 720 tcacgaacat gggcatggctgcggacccct cgtcatcagg tgcatagcaa gtgaaagcaa 780 gtgttcacaa cagtgaaaagttgagcgtca tttttcttag tgtgccaaga gttcgatgtt 840 agcgtttacg ttgtattttcttacactgtg tcattctgtt agatactaac atttcattga 900 tgacgaagac atacttaatcgatatttggt t 931 3 23 DNA Homo sapiens 3 cacacaggat ccatgaacgg aga 23 433 DNA Homo sapiens 4 cacacaaagc tttgagggga gttactcgtc atc 33 5 576 DNAHomo sapiens 5 atgaacggag acgacgcctt tgcaaggaga cccagggatg atgctcaaatatcagagaag 60 ttacgaaagg ccttcgatga tattgccaaa tacttctcta agaaagagtgggaaaagatg 120 aaatcctcgg agaaaatcgt ctatgtgtat atgaagctaa actatgaggtcatgactaaa 180 ctaggtttca aggtcaccct cccacctttc atgcgtagta aacgggctgcagacttccac 240 gggaatgatt ttggtaacga tcgaaaccac aggaatcagg ttgaacgtcctcagatgact 300 ttcggcagcc tccagagaat cttcccgaag atcatgccca agaagccagcagaggaagaa 360 aatggtttga aggaagtgcc agaggcatct ggcccacaaa atgatgggaaacagctgtgc 420 cccccgggaa atccaagtac cttggagaag attaacaaga catctggacccaaaaggggg 480 aaacatgcct ggacccacag actgcgtgag agaaagcagc tggtggtttatgaagagatc 540 agcgaccctg aggaagatga cgagtaactc ccctcg 576 6 576 DNAHomo sapiens 6 atgaacggag acgacgcctt tgtacggaga cctagggttg gttctcaaataccacagaag 60 atgcaaaagg ccttcgatga tattgccaaa tacttctctg agaaagagtgggaaaagatg 120 aaagcctcgg agaaaatcat ctatgtgtat atgaagagaa agtatgaggccatgactaaa 180 ctaggtttca aggccaccct cccacctttc atgcgtaata aacgggtcgcagacttccag 240 gggaatgatt ttgataatga ccctaaccgt gggaatcagg ttgaacatcctcagatgact 300 ttcggcaggc tccagggaat cttcccgaag atcacgcccg agaagccagcagaggaagga 360 aatgattcaa agggagtgcc agaagcatct ggcccacaga acaatgggaaacagctgcgc 420 ccctcaggaa aactaaatac ctctgagaag gttaacaaga catctggacccaaaaggggg 480 aaacatgcct ggacccacag agtgcgtgag agaaagcaac tggtggattatgaagagatc 540 agcgaccctg cggaagatga cgagtaactc ccctca 576 7 24 DNA Homosapiens 7 ctaaagccat gcagagaagg aagc 24 8 25 DNA Homo sapiens 8agatctctta ttaatcttc cagaaa 25 9 23 DNA Homo sapiens 9 gtgctcaaataccagagaag atc 23 10 23 DNA Homo sapiens 10 ttttgggtcc agatctcctc gtg 2311 24 DNA Homo sapiens 11 ggaagagtgg gaaaagatga aagt 24 12 22 DNA Homosapiens 12 ccccttttgg gtccagatat ca 22 13 25 DNA Homo sapiens 13aaatcgtcta tgtgtatatg aagct 25 14 22 DNA Homo sapiens 14 gggtcgctgatctcttcata ac 22 15 23 DNA Homo sapiens 15 gttctcaaat accacagaag atg 2316 20 DNA Homo sapiens 16 ctctgctggc ttctcgggcg 20 17 27 DNA Homosapiens 17 acagcattac caaggacagc agccacc 27 18 27 DNA Homo sapiens 18gccaacagca agatgcatac cagggac 27 19 9 PRT Homo sapiens 19 Arg Leu LeuGlu Phe Tyr Leu Ala Met 1 5 20 9 PRT Homo sapiens 20 Ser Leu Ala Gln AspAla Pro Pro Leu 1 5 21 9 PRT Homo sapiens 21 Ser Thr Leu Glu Lys Ile AsnLys Thr 1 5 22 9 PRT Homo sapiens 22 Lys Ala Ser Glu Lys Ile Ile Tyr Val1 5 23 9 PRT Homo sapiens 23 Asp Ala Phe Val Arg Arg Pro Arg Val 1 5 249 PRT Homo sapiens 24 Gln Ile Pro Gln Lys Met Gln Lys Ala 1 5 25 9 PRTHomo sapiens 25 Met Thr Lys Leu Gly Phe Lys Ala Thr 1 5 26 9 PRT Homosapiens 26 Met Thr Phe Gly Arg Leu Gln Gly Ile 1 5 27 9 PRT Homo sapiens27 Asn Thr Ser Glu Lys Val Asn Lys Thr 1 5 28 10 PRT Homo sapiens 28 TyrVal Tyr Met Lys Arg Lys Tyr Glu Ala 1 5 10 29 10 PRT Homo sapiens 29 TyrMet Lys Arg Lys Tyr Glu Ala Met Thr 1 5 10 30 10 PRT Homo sapiens 30 GluAla Met Thr Lys Leu Gly Phe Lys Ala 1 5 10 31 10 PRT Homo sapiens 31 MetThr Lys Leu Gly Phe Lys Ala Thr Leu 1 5 10 32 10 PRT Homo sapiens 32 ArgLeu Gln Gly Ile Gly Pro Lys Ile Thr 1 5 10 33 10 PRT Homo sapiens 33 GlnLeu Arg Pro Ser Gly Lys Leu Asn Thr 1 5 10 34 10 PRT Homo sapiens 34 GlyIle Phe Pro Lys Ile Thr Pro Glu Lys 1 5 10 35 10 PRT Homo sapiens 35 LysLeu Asn Thr Ser Glu Lys Val Asn Lys 1 5 10 36 10 PRT Homo sapiens 36 LysTyr Glu Ala Met Thr Lys Leu Gly Phe 1 5 10 37 9 PRT Homo sapiens 37 HisPro Gln Met Thr Phe Gly Arg Leu 1 5 38 9 PRT Homo sapiens 38 Gly Pro GlnAsn Asn Gly Lys Gln Leu 1 5 39 8 PRT Homo sapiens 39 Arg Val Arg Glu ArgLys Gln Leu 1 5 40 9 PRT Homo sapiens 40 Tyr Glu Ala Met Thr Lys Leu GlyPhe 1 5 41 9 PRT Homo sapiens 41 Arg Glu Arg Lys Gln Leu Val Ile Tyr 1 542 9 PRT Homo sapiens 42 Lys Gln Leu Val Ile Tyr Glu Glu Ile 1 5 43 10PRT Homo sapiens 43 Met Thr Phe Gly Arg Leu Gln Gly Ile Phe 1 5 10 44 9PRT Homo sapiens 44 Lys Ser Ser Glu Lys Ile Val Tyr Val 1 5 45 9 PRTHomo sapiens 45 Val Met Thr Lys Leu Gly Phe Lys Val 1 5 46 10 PRT Homosapiens 46 Tyr Val Tyr Met Lys Leu Asn Tyr Glu Val 1 5 10 47 10 PRT Homosapiens 47 Lys Leu Asn Tyr Glu Val Met Thr Lys Leu 1 5 10 48 9 PRT Homosapiens 48 Phe Ala Arg Arg Pro Arg Asp Asp Ala 1 5 49 9 PRT Homo sapiens49 Gln Ile Ser Glu Lys Leu Arg Lys Ala 1 5 50 9 PRT Homo sapiens 50 MetThr Phe Gly Ser Leu Gln Arg Ile 1 5 51 9 PRT Homo sapiens 51 Ser Leu GlnArg Ile Phe Pro Lys Ile 1 5 52 9 PRT Homo sapiens 52 Lys Ile Val Tyr ValTyr Met Lys Leu 1 5 53 9 PRT Homo sapiens 53 Lys Leu Arg Lys Ala Phe AspAsp Ile 1 5 54 10 PRT Homo sapiens 54 Lys Leu Arg Lys Ala Phe Asp AspIle Ala 1 5 10 55 10 PRT Homo sapiens 55 Tyr Met Lys Leu Asn Tyr Glu ValMet Thr 1 5 10 56 10 PRT Homo sapiens 56 Met Thr Lys Leu Gly Phe Lys ValThr Leu 1 5 10 57 10 PRT Homo sapiens 57 Gln Leu Cys Pro Pro Gly Asn ProSer Thr 1 5 10 58 9 PRT Homo sapiens 58 Lys Leu Asn Tyr Glu Val Met ThrLys 1 5 59 10 PRT Homo sapiens 59 Asn Tyr Glu Val Met Thr Lys Leu GlyPhe 1 5 10 60 9 PRT Homo sapiens 60 Arg Pro Gln Met Thr Phe Gly Ser Leu1 5 61 9 PRT Homo sapiens 61 Lys Pro Ala Glu Glu Glu Asn Gly Leu 1 5 629 PRT Homo sapiens 62 Gly Pro Gln Asn Asp Gly Lys Gln Leu 1 5 63 9 PRTHomo sapiens 63 Cys Pro Pro Gly Asn Pro Ser Thr Leu 1 5 64 8 PRT Homosapiens 64 Arg Leu Arg Glu Arg Lys Gln Leu 1 5 65 8 PRT Homo sapiens 65Arg Pro Arg Asp Asp Ala Gln Ile 1 5 66 9 PRT Homo sapiens 66 Lys Pro AlaGlu Glu Glu Asn Gly Leu 1 5 67 9 PRT Homo sapiens 67 Tyr Glu Val Met ThrLys Leu Gly Phe 1 5 68 9 PRT Homo sapiens 68 Arg Glu Arg Lys Gln Leu ValVal Tyr 1 5 69 9 PRT Homo sapiens 69 Lys Gln Leu Val Val Tyr Glu Glu Ile1 5 70 10 PRT Homo sapiens 70 Met Thr Phe Gly Ser Leu Gln Arg Ile Phe 15 10 71 9 PRT Homo sapiens 71 Lys Ile Gln Lys Ala Phe Asp Asp Ile 1 5 729 PRT Homo sapiens 72 Lys Ala Ser Glu Lys Ile Phe Tyr Val 1 5 73 9 PRTHomo sapiens 73 Ala Met Thr Lys Leu Gly Phe Lys Ala 1 5 74 9 PRT Homosapiens 74 Arg Leu Gln Gly Ile Ser Pro Lys Ile 1 5 75 9 PRT Homo sapiens75 Arg Leu Arg Glu Arg Lys Gln Leu Val 1 5 76 9 PRT Homo sapiens 76 AspAla Phe Ala Arg Arg Pro Thr Val 1 5 77 9 PRT Homo sapiens 77 Phe Ala ArgArg Pro Thr Val Gly Ala 1 5 78 9 PRT Homo sapiens 78 Gln Ile Pro Glu LysIle Gln Lys Ala 1 5 79 9 PRT Homo sapiens 79 Met Thr Phe Gly Arg Leu GlnGly Ile 1 5 80 9 PRT Homo sapiens 80 Glu Leu Cys Pro Pro Gly Lys Pro Thr1 5 81 10 PRT Homo sapiens 81 Tyr Val Tyr Met Lys Arg Lys Tyr Glu Ala 15 10 82 10 PRT Homo sapiens 82 Glu Ala Met Thr Lys Leu Gly Phe Lys Ala 15 10 83 10 PRT Homo sapiens 83 Met Thr Lys Leu Gly Phe Lys Ala Thr Leu 15 10 84 10 PRT Homo sapiens 84 Arg Ala Glu Asp Phe Gln Gly Asn Asp Leu 15 10 85 10 PRT Homo sapiens 85 Glu Leu Cys Pro Pro Gly Lys Pro Thr Thr 15 10 86 9 PRT Homo sapiens 86 Thr Leu Pro Pro Phe Met Cys Asn Lys 1 5 8710 PRT Homo sapiens 87 Lys Ile Phe Tyr Val Tyr Met Lys Arg Lys 1 5 10 8810 PRT Homo sapiens 88 Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe 1 5 10 899 PRT Homo sapiens 89 Arg Pro Gln Met Thr Phe Gly Arg Leu 1 5 90 9 PRTHomo sapiens 90 Gly Pro Gln Asn Asp Gly Lys Glu Leu 1 5 91 8 PRT Homosapiens 91 Arg Leu Arg Glu Arg Lys Gln Leu 1 5 92 9 PRT Homo sapiens 92Phe Ser Lys Glu Glu Trp Glu Lys Met 1 5 93 9 PRT Homo sapiens 93 Tyr GluAla Met Thr Lys Leu Gly Phe 1 5 94 9 PRT Homo sapiens 94 Arg Glu Arg LysGln Leu Val Ile Tyr 1 5 95 9 PRT Homo sapiens 95 Leu Gln Gly Ile Ser ProLys Ile Met 1 5 96 9 PRT Homo sapiens 96 Lys Gln Leu Val Ile Tyr Glu GluIle 1 5 97 9 PRT Homo sapiens 97 Ala Met Thr Lys Leu Gly Glu Lys Val 1 598 9 PRT Homo sapiens 98 Ala Met Thr Lys Leu Gly Phe Lys Val 1 5 99 9PRT Homo sapiens 99 Phe Ala Lys Arg Pro Arg Asp Asp Ala 1 5 100 9 PRTHomo sapiens 100 Lys Ala Ser Glu Lys Arg Ser Lys Ala 1 5 101 10 PRT Homosapiens 101 Tyr Val Tyr Met Lys Arg Asn Tyr Lys Ala 1 5 10 102 10 PRTHomo sapiens 102 Lys Ala Met Thr Lys Leu Gly Phe Lys Val 1 5 10 103 9PRT Homo sapiens 103 Met Thr Lys Leu Gly Phe Lys Val Thr 1 5 104 10 PRTHomo sapiens 104 Met Thr Lys Leu Gly Phe Lys Val Thr Leu 1 5 10 105 10PRT Homo sapiens 105 Arg Ile Gln Val Glu His Pro Gln Met Thr 1 5 10 1069 PRT Homo sapiens 106 Met Thr Phe Gly Arg Leu His Arg Ile 1 5 107 9 PRTHomo sapiens 107 Thr Leu Pro Pro Phe Met Cys Asn Lys 1 5 108 10 PRT Homosapiens 108 Asn Tyr Lys Ala Met Thr Lys Leu Gly Phe 1 5 10 109 9 PRTHomo sapiens 109 His Pro Gln Met Thr Phe Gly Arg Leu 1 5 110 9 PRT Homosapiens 110 Gly Pro Gln Asn Asp Gly Lys Gln Leu 1 5 111 8 PRT Homosapiens 111 Arg Leu Arg Glu Arg Lys Gln Leu 1 5 112 9 PRT Homo sapiens112 Arg Glu Arg Lys Gln Leu Val Ile Tyr 1 5 113 9 PRT Homo sapiens 113Lys Gln Leu Val Ile Tyr Glu Glu Ile 1 5 114 10 PRT Homo sapiens 114 MetThr Phe Gly Arg Leu His Arg Ile Ile 1 5 10 115 9 PRT Homo sapiens 115Ser Ile Ser Ser Cys Leu Gln Gln Leu 1 5 116 9 PRT Homo sapiens 116 GlyThr Gly Gly Ser Thr Gly Asp Ala 1 5 117 9 PRT Homo sapiens 117 Arg AlaSer Gly Pro Gly Gly Gly Ala 1 5 118 9 PRT Homo sapiens 118 Gly Ala ArgGly Pro Glu Ser Arg Leu 1 5 119 9 PRT Homo sapiens 119 Ala Thr Pro MetGlu Ala Glu Leu Ala 1 5 120 9 PRT Homo sapiens 120 Phe Thr Val Ser GlyAsn Ile Leu Thr 1 5 121 9 PRT Homo sapiens 121 Leu Thr Ala Ala Asp HisArg Gln Leu 1 5 122 9 PRT Homo sapiens 122 Gln Leu Ser Leu Leu Met TrpIle Thr 1 5 123 9 PRT Homo sapiens 123 Leu Met Trp Ile Thr Gln Cys PheLeu 1 5 124 9 PRT Homo sapiens 124 Phe Ala Thr Pro Met Glu Ala Glu Leu 15 125 9 PRT Homo sapiens 125 Thr Val Ser Gly Asn Ile Leu Thr Ile 1 5 12610 PRT Homo sapiens 126 Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 1 5 10127 10 PRT Homo sapiens 127 Gly Ala Pro Arg Gly Pro His Gly Gly Ala 1 510 128 10 PRT Homo sapiens 128 Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala 15 10 129 10 PRT Homo sapiens 129 Ile Thr Gln Cys Phe Leu Pro Val Phe Leu1 5 10

1. An isolated peptide consisting of 9 to 20 amino acids, comprising thesequence of ArgLeuLeuGluPheTyrLeuAlaMet (SEQ ID NO: 19) wherein saidisolated peptide binds to an HLA molecule to form a complex.
 2. Theisolated peptide of claim 1, wherein said complex stimulatesproliferation of cytolytic T lymphocytes.
 3. The isolated peptide ofclaim 1, wherein said HLA molecule is an HLA-A2 molecule.
 4. Theisolated peptide of claim 3, wherein said HLA molecule is HLA-A*0201. 5.A method for stimulating proliferation of cytolytic T lymphocytescomprising combining a cell which presents an MHC or HLA molecule on itssurface with the isolated peptide of claim 1, and a T lymphocytecontaining sample, under conditions favoring formation of a complexbetween said HLA molecule and said peptide, and proliferation ofcytolytic T lymphocytes specific for said complex.
 6. The method ofclaim 5, wherein said HLA molecule is HLA-A2.
 7. The method of claim 5,comprising administering said peptide to a subject in need ofstimulation of cytolytic T lymphocytes.
 8. The method of claim 7,wherein said peptide is administered with an adjuvant.
 9. The method ofclaim 7, wherein said peptide is administered with an interleukin. 10.The method of claim 7, wherein said subject has a neoplastic disorder.11. The method of claim 10, wherein said neoplastic disorder ismelanoma.
 12. The method of claim 5, comprising combining said cell,said peptide, and said T lymphocyte containing sample in vitro. 13.Isolated cytolytic T lymphocyte which specifically recognizes a complexof an MHC or HLA molecule and the isolated peptide of claim
 1. 14. Theisolated cytolytic T lymphocyte of claim 13, wherein said HLA moleculeis HLA-A2.
 15. A monoclonal antibody that specifically bind the peptideof claim 1 in complex with a carrier protein.
 16. The monoclonalantibody of claim 15 wherein said antibody is humanized.
 17. Themonoclonal antibody of claim 15 wherein said carrier protein is an HLAmolecule.
 18. A purified antisera that specifically bind the peptide ofclaim 1 in complex with an HLA molecule.
 19. The antisera of claim 16which is purified by antigen affinity column.
 20. Composition of mattercomprising at least one peptide, the amino acid sequence of which is setforth at SEQ ID NO: 1, and a pharmaceutically acceptable carrier.
 21. Anisolated nucleic acid molecule with a sequence which encodes a peptideof claim
 1. 22. Expression vector comprising the isolated nucleic acidmolecule of claim 21, operably linked to a promoter.
 23. The expressionvector of claim 22 wherein said promoter is an inducible promoter. 24.The expression vector of claim 22 wherein said promoter is an eukaryoticconstitutive promoter.
 25. Recombinant cell comprising the isolatednucleic acid molecule of claim
 21. 26. The recombinant cell of claim 25,further comprising a recombinant nucleic acid molecule which encodes anHLA molecule.
 27. A polytope comprising a plurality of amino acidsequences which correspond to peptides which bind to MHC molecules,wherein at least one of said amino acid sequence is the amino acidsequence of SEQ ID NO:
 19. 28. An isolated nucleic acid molecule with asequence which encodes a polytope of claim
 27. 29. Expression vectorcomprising the isolated nucleic acid molecule of claim 28, operablylinked to a promoter.
 30. The expression vector of claim 29 wherein saidpromoter is an inducible promoter.
 31. The expression vector of claim 29wherein said promoter is an eukaryotic constitutive promoter. 32.Recombinant cell comprising the isolated nucleic acid molecule of claim28.
 33. The recombinant cell of claim 32, further comprising arecombinant nucleic acid molecule which encodes an HLA molecule.